Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay

The potential of the test material to cause mutagenic effects in bacteria was assessed. Based on the results of this assay, the test material was not mutagenic in any strain of Salmonella typhimurium tested.

Chromosome Aberration

The potential of the test material to induce structural chromosomal aberrations was determined. Under the conditions of the study, the test material was considered not to induce any statistically significant increases in the frequency of cells with aberrations and therefore was considered to be non-clastogenic.

Mouse Lymphoma Assay

A study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. Under the conditions of this study the test material is not mutagenic in the presence and absence of metabolic activation.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Ames Test

The potential of the test material to cause mutagenic effects in five selected strains of Salmonella typhimurium was assessed in accordance with the standardised guideline OECD 471 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was diluted in acetone and tested in both an initial and repeat assay. Based on the results of the range-finding test, test concentrations of 125, 250, 500, 1 000, and 2 000 µg/plate without metabolic activation (-S9) and 250, 500, 1 000, 2 000, and 4 000 µg/plate with metabolic activation (+ S9) were selected for the initial mutagenicity assay. Based on the results of the initial assay, dose intervals of 500, 1 000, 1 500, 2 000 and 3 000 µg/plate (-S9) and 500, 1 000, 2 000, 3 000 and 4 000 µg/plate (+ S9) were selected for the repeat assay to confirm the negative response demonstrated in the initial assay. In both study phases, a 100 µL sample of each concentration was tested with and without metabolic activation (rat liver S9 mix). Two positive controls and two vehicle controls (acetone and DMSO) were tested concurrently for each strain. The vehicle controls were tested using 100 µL samples for both DMSO and acetone.

In both the initial and repeat assays, the test material did not induce a significant increase in revertant colonies (equal to or greater than three times the acetone controls) in tester strains TA98, TA100, TA1535, TA1537 or TA1538 at any dose level tested, either with or without metabolic activation.

Toxicity, as indicated by a >50 % reduction in the mean number of revertant colonies when compared to the vehicle control, was observed in the initial assay at 2 000 µg/plate in TA100 (-S9) and TA1537 (+S9), and was observed in the repeat assay at 2 000 µg/plate in TA1537 (+S9) and at 3 000 µg/plate in TA1538 (-S9) and TA1537 (+S9).

Based on the results of this assay, the test material was not mutagenic in any strain of Salmonella typhimurium tested.

Chromosome Aberration

The potential of the test material to induce structural chromosomal aberrations was determined in a GLP study which was conducted in accordance with standardised guidelines OECD 473 and Japanese Guidelines for Screening Mutagenicity Testing Of Chemicals. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated; 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2 % final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation.

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by precipitate. The dose levels selected were 0, 2.5, 5, 10, 20, 40 and 80 µg/mL for 4 hour exposure with and without S9 and 24-hour without S9.

All vehicle (acetone) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control materials induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material was toxic at the recommended dose level but did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the lowest precipitating dose level.

Under the conditions of the study, the test material was considered not to induce any statistically significant increases in the frequency of cells with aberrations and, therefore was considered to be non-clastogenic.

Mouse Lymphoma Assay

A study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The study was conducted in accordance with the standardised guidelines OECD 490, EU Method B.17 and EPA OPPTS 870.5300 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

One main mutagenicity test was performed. In the test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at eight dose levels in duplicate, together with vehicle (acetone), and positive controls using 4 hour exposure groups both in the absence and presence of metabolic activation (2 % S9), and a 24 hour exposure group in the absence of metabolic activation

The dose range of test material used in the main test was selected following the results of a preliminary toxicity test.The dose levels plated for viability and expression of mutant colonies were 2.5, 5, 10, 20, 30, 40 µg/mL for 4-hour treatment without S9, 4-hour treatment with S9 (2 %) and 24-hour treatment without S9.

The maximum dose level used in the Mutagenicity Test was limited by the presence of precipitate of the test material. Precipitate of the test material was observed at 30 and 40 µg/mL in the 4-hour exposure groups, and at 40 µg/mL in the 24-hour exposure group, at the end of the exposure period in the Mutagenicity Test. The vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.

The test material did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups.

Under the conditions of this study the test material is not mutagenic in the presence and absence of metabolic activation.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the substance does not require classification with respect to genetic toxicity.